Stationary phase is the heart of chromatographic separation process. Due to the absence of suitable common stationary phase immobilization technique, gas chromatography and liquid chromatography utilize completely two different types of materials as the separation media: polymeric stationary phases for gas chromatography and monomeric stationary phases for liquid chromatography. Immobilization of stationary phase in gas chromatography is relatively simple and straightforward and generally involves free radical cross-linking or chemical bonding technique. Unlike gas chromatography, stationary phase in liquid chromatography makes direct contact with the mobile phase in its liquid state, and hence requires strong chemical anchorage with the substrate to prevent from washing away with the mobile phase.
In order to prevent from losing the stationary phase by dissolving in the mobile phase, reversed phase and normal phase high-performance liquid chromatography (RP-HPLC, NP-HPLC) were evolved with exclusively different combinations of stationary phase and mobile phase. RP-HPLC utilizes non-polar stationary phases and polar mobile phases, whereas NP-HPLC utilizes polar stationary phases and non-polar mobile phases. This artificial classification and exclusivity in stationary phase/mobile phase selection substantially limit the power of high-performance liquid chromatography. As such, a limited number of monomeric nonpolar stationary phases including C4, C8, C18, and C30 are used in RP-HPLC and diol, cyano, and amino polar monomeric ligands are used in NP-HPLC.
The strict restrictions in selecting stationary phase/mobile phase combination has been relaxed to some extent after the introduction of bonded phases where monomeric entities are chemically bonded to the surface silanol groups present on silica substrate via silane chemistry. This approach of creating bonded phases has undoubtedly improved column performance, increased pH stability and prolonged its lifetime. The advent of the bonded phases led to a new direction in liquid chromatography known as hydrophilic interaction chromatography (HILIC). HILIC is a hybrid liquid phase separation approach that combines, to a great extent, reversed phase and normal phase liquid chromatography. HILIC expands the separation power of liquid chromatography towards highly polar analyte(s) that can't be dealt in normal phase chromatography due to the restricted choice of organic solvents (e.g., hexane, isooctane, carbon tetrachloride) in which many of the polar analytes are barely soluble. HILIC employs a polar stationary phase, such as silica, diol, amino, and cyano phases that are used for normal phase liquid chromatography and uses polar organic or organo-aqueous solvent system as the mobile phase, such as those used in as in reversed phase liquid chromatography. Recently, introduction of bonded phases, such as diol, cyano, and amino ligands by bonding to a silica substrate via silane chemistry has allowed polar organic or organo-aqueous mobile phases to be used in hydrophilic interaction chromatography. Unfortunately, due to relatively weak bonding between the silica substrate and the organic ligands in these “so called” bonded phases, phase-bleeding often occurs upon exposure to polar organic solvents or organo-aqueous solvent mobile phases, resulting in a continuous shift in chromatographic retention and selectivity change due to the exposure of free surface silanol groups of the silica support.
Supports for reversed phase high-performance liquid chromatography (RP-HPLC), normal phase high-performance liquid chromatography (NP-HPLC) or HILIC are silica particles coated with organic ligands via silane chemistry. As a result, only a very small portion of the stationary phase contributes to retention and selectivity. The current bonded phases in RP-HPLC, NP-HPLC, and HILIC suffer from a number of shortcomings: silica particles limit the loading of organic ligands; silane chemical bonding of target organic ligands to the substrate surface steric limits incorporation of organic ligands leaving many surface silanol groups on the silica surface; and low carbon loading of organic ligands results in chemical instability, particularly using basic solvents.
It is evident that regardless of the separation mode used in high-performance liquid chromatography, all stationary phases are based on monomeric ligands with limited intermolecular interaction capability extended towards the analytes. For example, reversed phase stationary phases interact with the analytes with weak London dispersion forces and normal phases use limited dipole-dipole interaction/hydrogen bonding. Maximum separation/extraction potential of HPLC can only be utilized when all possible intermolecular interactions including London dispersion, dipole-dipole interaction, hydrogen bonding and π-π stacking interactions are exercised.
Unlike high-performance liquid chromatography, solid phase extraction utilized silica particles coated with similar organic ligands as seen in HPLC stationary phases and therefore the choices are limited. Chemical incorporation of various organic polymers into the sol-gel hybrid inorganic-organic matrix would open up the possibility of creating hundreds of novel sorbents with unique selectivity.
Organic polymers/macromeres/dendrimers/biopolymers represent a class of compounds possessing versatile surface chemistry with unique and rich functionality. A successful immobilization technique such as sol-gel synthesis can chemically incorporate these polymers in a 3D network of metal oxides e.g., silica, germania, titania backbone. All gas chromatographic stationary phases including polysiloxanes, polyethylene glycols, phenylpolycarborane-siloxanes as well as other polymers not being used as gas chromatographic stationary phases can be readily used as liquid chromatographic stationary phases if they possess at least one terminal hydroxyl functional group. The effective incorporation of flexible organic polymer into rugged inorganic polymeric network may dramatically and synergistically expand the separation power of HPLC as well as the extraction efficiency when used as solid phase extraction sorbents.
Stationary phases for RP-HPLC, NP-HPLC, and HILIC that overcome the inherent shortcomings of the current stationary phase manufacturing technology is the focus of the invention, where a sol-gel synthesis generates the support and the organic functionality simultaneously. Organic units can be included as hydroxyl terminated monomers and polymers, and included by condensation with the sol-precursors, sol, or gel. A method of preparation that separately promotes hydrolysis of sol-precursors to the sol and condensation of the sol to the gel is carried out. Due to the strong chemical bonding between the 3D network of metal oxide and organic monomer/polymer/macromere/dendrimer, the invention opens up the possibility of eliminating artificial classification of reversed phase and normal phase high-performance liquid chromatography into a unified high-performance liquid chromatography that can use any polymeric stationary phase of desired polarity in combination with any organic solvent (nonpolar, medium polar, polar) or organo-aqueous solvent as the mobile phase to achieve the desired separation goal. In addition, the new approach will allow scientists to exploit high chemical stability (pH 1-13) and thermal stability of the sol-gel polymeric stationary phases and solid phase sorbents to maximize the separation potential as well as the extraction efficiency.